The worldwide emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) is severely complicating the current tuberculosis (TB) epidemic. New TB drugs are urgently needed to combat MDR/XDR TB and to improve the current 6-month drug regimens for non-resistant TB. The folate biosynthetic pathway has been an attractive target for antibiotic development since it is absent in humans. A preliminary study in our laboratory using a transposon insertion library in M. smegmatis has identified several novel determinants of antifolate resistance in mycobacteria. One antifolate sensitive mutant encodes a homolog of the eukaryotic-type protein kinase G (PknG), recently identified as possible regulator of persistence of pathogenic mycobacteria in macrophages. Preliminary studies reveal that PknG regulates de novo folate biosynthesis by modulating the activity of a dihydroneopterin triphosphate pyrophosphatase that controls the influx of pterin moiety into the folate pathway. This novel regulatory mechanism has not been previously identified for de novo folate biosynthesis. Both genetic interruption and specific chemical inhibition of PknG kinase activity result in hyper-susceptibility of mycobacteria not only to antifolate drugs but also other antibiotics, including frontline TB drugs such as rifampicin and ethambutol. This is due to a direct effect on de novo folate biosynthesis and an indirect effect by altering cell wall permeability, respectively. The central hypothesis of this application is that genes defining intrinsic antifolate resistance encode proteins that can be targeted by potentiators that sensitize Mtb to antifolate drugs by inhibiting the resistance mechanisms. Specifically, pharmaceutical inactivation of PknG could sensitize Mtb to antifolates and multiple other approved drugs, to which it is currently resistant. Three specific aims are designed to test this hypothesis. First, using a non-biased approach, we will identify and characterize the entire genome-wide antifolate resistant determinants (the antifolate resistome) of Mtb. Secondly, we will rigorously investigate the molecular mechanisms of PknG-regulated folate-biosynthesis in Mtb. Lastly, we will characterize the potentiating effects of PknG inhibitors on antifolate drugs and the efficacy of their combined effect against drug-resistant and non-resistant Mtb. These proposed studies will not only provide insight into a previously unknown regulatory mechanism of de novo folate biosynthesis in bacteria but also into the mechanisms of intrinsic resistance of Mtb to antifolate drugs. In terms of drug development, these studies will reveal novel targets and provide proof of concept that inhibition of intrinsic resistance pathways in Mtb can be used to improve the effectiveness of already available antibiotics. )